Masako Nishizuka

Sexual dimorphism in synaptic organization in the amygdala and its dependence on neonatal hormone environment.

Abstract Electron microscopic observation was made on coronal sections from the middle part of the medial amygdaloid nucleus (AMN) of the rats. According to postsynaptic elements, shaft synapses (SHS) terminating on dendritic shafts, spine synapses (SPS) made on dendritic spines and somatic synapses (SOS) made on neuronal cell bodies were differentially counted on a field of 10,000 sq. μm in a single section of the identical level of the nucleus in each brain. The total number of SHS, SPS, and SOS per 10,000 sq. μm in normal adult male rats was significantly larger than in normal female rats. This sex difference in synaptic number was due to a significant increase in number of SHS in normal male rats, while the incidences of SPS and SOS in normal males were not significantly different from those in normal females. A single injection of 1.25 mg testosterone propionate (TP) to day 5 females caused a marked increase in the number of SHS, compared to normal females. The incidence of SHS in androgenized females was almost the same as that in normal males. However, the other two components of synaptic population (SPS and SOS) were not influenced by neonatal treatment with TP. On the other hand, neonatal orchidectomy resulted in a significant decrease in the number of SHS to the level comparable to that of normal females. These results clearly indicate the existence of sexual dimorphism in the synaptic pattern in the AMN. Furthermore, this sex difference was highly dependent on the neonatal presence of androgen, which effectively stimulated shaft synaptogenesis during early postnatal period. The modification of synaptic patterns caused by neonatal exposure to androgen in the amygdala may participate in the possible process of sexual differentiation of neuroendocrine and behavioral parameters of the amygdaloid function.

Organizational action of estrogen on synaptic pattern in the amygdala: implications for sexual differentiation of the brain

Estrogen markedly promotes the specific formation of dendritic shaft synapses in the medial amygdaloid nucleus (AMN) of the rat when given in early postnatal days. The high incidence of this type of synapse permanently persists until sacrifice at adult ages. The data provide evidence for the synaptic plasticity of the developing amygdala to estrogen and suggest a possible morphological mechanism for sexual differentiation of the brain.

Sex differences in the anteroventral periventricular nucleus of the preoptic area and in the related effects of androgen in prenatal rats

The density of cells in the anteroventral periventricular nucleus (AVPv) of the preoptic area of female rats was greater than that of males on day 21 of gestation. It appears that this difference between the sexes is caused by the action of androgen since the density of cells in the AVPv of female fetuses fell to the density in males when the mother received injections of testosterone propionate (TP) on days 14-18 of gestation. Pycnotic cells were more frequently found in oil-treated control males and TP-treated female fetuses than in control female fetuses. This result suggests that the prenatal injections of TP enhanced the rate of degeneration of the cells in the AVPv.

Androgen Enhances Neuronal Degeneration in the Developing Preoptic Area: Apoptosis in the Anteroventral Periventricular Nucleus (AVPvN-POA)

Perinatal treatment of female rats with androgen decreases the nuclear volume of the anteroventral periventricular nucleus of the preoptic area (AVPvN-POA). In order to examine the effect of androgen on neurogenesis, bromodeoxyuridine (BrdU) was given once on Day 15 of gestation (= E15) to pregnant rats that also received testosterone propionate (TP) injections. When examined at E17, the number of BrdU-labeled neurons in the AVPvN-POA was not significantly different among control female, male, and androgenized female fetuses, suggesting that androgen does not interfere with neurogenesis. At E21, a significant reduction of BrdU-labeled AVPvN neurons was observed in males and androgenized females. These findings support the hypothesis that elimination of a population by cell death is enhanced in males and androgenized females. Similar selective elimination of the AVPvN neurons occurred in the female following neonatal TP treatment. In order to investigate the nature of androgen-induced cell death in the AVPvN-POA, specific labeling of nuclear DNA fragmentation was performed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. The number of TUNEL-positive cells was significantly greater in neonatally androgenized females, compared to that in control females. Since DNA fragmentation is considered the most characteristic feature of apoptosis, and TUNEL method is based on direct, specific labeling of DNA fragmentation in nuclei in situ, the neuronal death in the AVPvN-POA is apoptotic, and perinatal androgen may induce the selective apoptotic cell death in the AVPvN-POA.

Occurrence of a N-terminal proteolytic fragment of neurocan, not a C-terminal half, in a perineuronal net in the adult rat cerebrum

Neurocan is a nervous tissue-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. In the adult rat brain, neurocan is completely cleaved into some proteoglycan fragments including the C-terminal half known as neurocan-C and a N-terminal fragment with a 130 kDa core glycoprotein (neurocan-130). We describe here the differential distribution of these two neurocan-derived CSPGs in the adult rat cerebrum and the occurrence of neurocan-130 as a new member of a perineuronal net-constituting molecule. At the light microscopic level, neurocan-130 exhibited pericellular localization around a subset of neurons in addition to diffuse distribution in the neuropil. In contrast, neurocan-C was distributed only diffusely in the neuropil. Double staining with anti-neurocan-130 and anti-synaptophysin antibodies suggested that neurocan-130 was localized in the vicinity of the synapses, but not at the synapses. Immunoelectron microscopy showed that neurocan-130 was mainly localized in the cytoplasm of glial cell processes, the so-called glial perineuronal net, encompassing the cell bodies of certain neurons. The presence of neurocan-130 in a limited number of glial cells may reflect some functional heterogeneity of the glia.

Age- and sex-related differences in the nerve growth factor distribution in the rat brain.

Levels of the nerve growth factor (NGF) have been measured in various brain regions of young and aged male and female rats of Wistar strain by means of a highly sensitive two-site enzyme immunoassay system for beta-NGF. Among the ten regions examined, the amount of NGF per wet weight of tissue was found to be highest in the hippocampus, irrespective of the sex and age. The NGF concentration in the hippocampus of female rats at 3 months of age was comparable to that of same aged males. Further, there was no significant difference in the NGF levels of the hippocampus between young and age males. However, the NGF level was significantly lower in aged females as compared to that in 3- or 4-month-old females, and hence the marked male-female difference was found in the NGF levels in aged Wistar rats.

Synaptogenesis and Neuronal Plasticity to Gonadal Steroids: Implications for the Development of Sexual Dimorphism in the Neuroendocrine Brain

Sex differences in adult neuroendocrine and behavioral responses largely depend on differences in the organization of the brain. Many of these are brought about by the exposure of undifferentiated brain to perinatal gonadal hormones, especially aromatizable androgen or estrogen (Goy and McEwen 1980; MacLusky and Naftolin 1981; Arai et al. 1983). Recent studies indicate that sex steroid hormones modulate and promote neuronal maturation and neurite growth in certain brain regions (Toran-Allerand 1984). Synaptogenesis can also be facilitated by estrogenic action in the hypothalamic arcuate nucleus (ARCN) and medial amygdaloid nucleus (MAN) during early postnatal development (Arai 1981). These organizational effects of gonadal steroids appear to be regionally specific and correlated with the presence and topographic localization of the sex steroid receptor-containing neurons (Goy and McEwen 1980).

Loss of dendritic synapses in the medial amygdala associated with kindling

Kindling stimulation was given in the basolateral amygdala (BLA), the septal area or the corpus callosum in the right hemisphere of adult rats. The density of dendritic synapses was electron microscopically studied in the medial amygdaloid nucleus (MAN) ipsi- and contralateral to the stimulation side. The number of dendritic synapses was markedly decreased in both sides of the MAN of 3 groups of kindled rats. Such reduction occurred in both dendritic shaft and spine synapses. The most remarkable decrease was obtained in the BLA kindling. These results suggest that a decrease of synapses may provide a morphological basis for kindling.

Cell surface-associated extracellular distribution of a neural proteoglycan, 6B4 proteoglycan/phosphacan, in the olfactory epithelium, olfactory nerve, and cells migrating along the olfactory nerve in chick embryos.

The immunocytochemical and immuno-electron microscopic distribution of a neural proteoglycan (PG) was investigated with a monoclonal antibody, MAb 6B4, in the olfactory epithelium, the olfactory nerve, and the cells originating the epithelium and migrating along the olfactory nerve toward the forebrain in chick embryos. The PG recognized by MAb 6B4, that is 6B4 PG, in the brain of early postnatal rats, is identical to phosphacan. In chick embryos, immunoreactivity to 6B4 PG appeared on embryonic day (ED) 3-3.5 in a thin layer beneath the olfactory epithelium. It disappeared immediately, then becoming apparent in the bundles of the olfactory nerve. The immunoreactivity in the nerve bundles gradually increased during ED 5-11. On the other hand, cell surface-associated extracellular localization of the immunoreactivity was seen in the olfactory epithelium on ED 6 and afterwards. Immunofluorescent double-labeling of 6B4 PG and gonadotropin-releasing hormone (GnRH) revealed that the cell bodies of both GnRH-containing cells and other cells migrating along the olfactory nerve were surrounded by a rim immunoreactive to the PG. Under an electron microscope, the surfaces of the cell bodies and of the neurites in the nerve bundles were surrounded by deposits immunoreactive to 6B4 PG. These results indicate that 6B4 PG in chick embryos is one type of cell surface-associated extracellular matrix molecule, and that 6B4 PG covered the surfaces of migrating cells and of elongating olfactory nerve. The cell surface-associated extracellular localization of 6B4 PG found in the nasal region, taken together with the binding properties of this PG with cell adhesion molecules shown in rat brains, suggested that 6B4 PG played a role in guiding the migration of cells along the olfactory nerve in chick embryos.

Changes in the amounts of chondroitin sulfate proteoglycans in rat brain after neonatal hypoxia-ischemia

Chondroitin sulfate proteoglycans have been shown to participate in the pathogenesis of neuronal damages in the injured adult central nervous system (CNS). Upregulated expression of chondroitin sulfate proteoglycans has been reported around the injured sites and depletion of these chondroitin sulfate proteoglycans brings about increased axonal regeneration in the injured adult CNS. To examine if chondroitin sulfate proteoglycans are also involved in the pathologic process of hypoxia‐ischemia in the neonatal brain, expressions of three chondroitin sulfate proteoglycans, neurocan, phosphacan, and neuroglycan C, were examined in rat brains after neonatal hypoxia‐ischemia. Hypoxic‐ischemic rats were produced by ligating the right carotid artery of 7‐day‐old rats, followed by 8% oxygen exposure. Western blot analysis revealed that in contrast to injured adult CNS, the amount of neurocan was reduced 24 hr after hypoxia in the neonatal hypoxic‐ischemic cerebral hemisphere. The amounts of phosphacan and neuroglycan C were also reduced significantly 24 hr after hypoxia at the right injured cortex compared to those at the left cortex. Surprisingly, the immunohistologic staining for phosphacan was conversely intensified both at 24 hr and 8 days after hypoxia at the infarcted area. In addition, the habenula and fascicules retroflexus in the right cerebral hemisphere degenerated and became intensely immunostained with the anti‐phosphacan antibody shortly after hypoxia. Hypoxic‐ischemic insult may unmask phosphacan epitopes at the injured sites, resulting in intensified immunostaining. Because intensified immunostaining for neurocan and neuroglycan C was not observed, unmasking seems to be specific to phosphacan among these three chondroitin sulfate proteoglycans.